PP1 Phosphatase Complexes: Undruggable No Longer.

The identification of inhibitors targeting regulatory subunits of serine/threonine PP1 phosphatases reported by Krzyzosiak et al. is a significant step in expanding the pharmacological regulation of phosphorylation beyond kinases. The selective inhibitor of the R15B phosphatase regulatory subunit, termed Raphin1, protects cells from stress and delays neurodegeneration in a mouse model of Huntington's disease.

CCDC86 is a novel Ki-67-interacting protein important for cell division.

The chromosome periphery is a network of proteins and RNAs that coats the outer surface of mitotic chromosomes. Despite the identification of new components, the functions of this complex compartment are poorly characterised. In this study, we identified a novel chromosome periphery-associated protein, CCDC86 (also known as cyclon). Using a combination of RNA interference, microscopy and biochemistry, we studied the functions of CCDC86 in mitosis. CCDC86 depletion resulted in partial disorganisation of the chromosome periphery with alterations in the localisation of Ki-67 (also known as MKI67) and nucleolin (NCL), and the formation of abnormal cytoplasmic aggregates. Furthermore, CCDC86-depleted cells displayed errors in chromosome alignment, altered spindle length and increased apoptosis. These results suggest that, within the chromosome periphery, different subcomplexes that include CCDC86, nucleolin and B23 (nucleophosmin or NPM1) are required for mitotic spindle regulation and correct kinetochore-microtubule attachments, thus contributing to chromosome segregation in mitosis. Moreover, we identified CCDC86 as a MYCN-regulated gene, the expression levels of which represent a powerful marker for prognostic outcomes in neuroblastoma.

Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis.

Compaction of chromosomes is essential for accurate segregation of the genome during mitosis. In vertebrates, two condensin complexes ensure timely chromosome condensation, sister chromatid disentanglement, and maintenance of mitotic chromosome structure. Here, we report that biallelic mutations in NCAPD2, NCAPH, or NCAPD3, encoding subunits of these complexes, cause microcephaly. In addition, hypomorphic Ncaph2 mice have significantly reduced brain size, with frequent anaphase chromatin bridge formation observed in apical neural progenitors during neurogenesis. Such DNA bridges also arise in condensin-deficient patient cells, where they are the consequence of failed sister chromatid disentanglement during chromosome compaction. This results in chromosome segregation errors, leading to micronucleus formation and increased aneuploidy in daughter cells. These findings establish "condensinopathies" as microcephalic disorders, with decatenation failure as an additional disease mechanism for microcephaly, implicating mitotic chromosome condensation as a key process ensuring mammalian cerebral cortex size.

Back to the new beginning: Mitotic exit in space and time.

The ultimate goal of cell division is to generate two identical daughter cells that resemble the mother cell from which they derived. Once all the proper attachments to the spindle have occurred, the chromosomes have aligned at the metaphase plate and the spindle assembly checkpoint (a surveillance mechanism that halts cells form progressing in the cell cycle in case of spindle - microtubule attachment errors) has been satisfied, mitotic exit will occur. Mitotic exit has the purpose of completing the separation of the genomic material but also to rebuild the cellular structures necessary for the new cell cycle. This stage of mitosis received little attention until a decade ago, therefore our knowledge is much patchier than the molecular details we now have for the early stages of mitosis. However, it is emerging that mitotic exit is not just the simple reverse of mitotic entry and it is highly regulated in space and time. In this review I will discuss the main advances in the field that provided us with a better understanding on the key role of protein phosphorylation/de-phosphorylation in this transition together with the concept of their spatial regulation. As this field is much younger, I will highlight general consensus, contrasting views together with the outstanding questions awaiting for answers.

Non-redundant functions of H2A.Z.1 and H2A.Z.2 in chromosome segregation and cell cycle progression.

H2A.Z is a H2A-type histone variant essential for many aspects of cell biology, ranging from gene expression to genome stability. From deuterostomes, H2A.Z evolved into two paralogues, H2A.Z.1 and H2A.Z.2, that differ by only three amino acids and are encoded by different genes (H2AFZ and H2AFV, respectively). Despite the importance of this histone variant in development and cellular homeostasis, very little is known about the individual functions of each paralogue in mammals. Here, we have investigated the distinct roles of the two paralogues in cell cycle regulation and unveiled non-redundant functions for H2A.Z.1 and H2A.Z.2 in cell division. Our findings show that H2A.Z.1 regulates the expression of cell cycle genes such as Myc and Ki-67 and its depletion leads to a G1 arrest and cellular senescence. On the contrary, H2A.Z.2, in a transcription-independent manner, is essential for centromere integrity and sister chromatid cohesion regulation, thus playing a key role in chromosome segregation.

Ki-67: More Hidden behind a 'Classic Proliferation Marker'.

Nuclear antigen Ki-67 is widely accepted as a cell proliferation marker in both research and cancer diagnostic settings. Despite its extensive use and clinical value, very little is still known about the biological function of Ki-67. A recent work published in Cell Reports has revealed important novel aspects of Ki-67 regulation that could provide new and extended prognostic and therapeutic value.

Chromosome clustering in mitosis by the nuclear protein Ki-67.

Ki-67 is highly expressed in proliferating cells, a characteristic that made the protein a very important proliferation marker widely used in the clinic. However, the molecular functions and properties of Ki-67 remained quite obscure for a long time. Only recently important discoveries have shed some light on its function and shown that Ki-67 has a major role in the formation of mitotic chromosome periphery compartment, it is associated with protein phosphatase one (PP1) and regulates chromatin function in interphase and mitosis. In this review, we discuss the role of Ki-67 during cell division. Specifically, we focus on the importance of Ki-67 in chromosome individualisation at mitotic entry (prometaphase) and its contribution to chromosome clustering and nuclear remodelling during mitotic exit.

Protein phosphatases at the nuclear envelope.

The nuclear envelope (NE) is a unique topological structure formed by lipid membranes (Inner and Outer Membrane: IM and OM) interrupted by open channels (Nuclear Pore complexes). Besides its well-established structural role in providing a physical separation between the genome and the cytoplasm and regulating the exchanges between the two cellular compartments, it has become quite evident in recent years that the NE also represents a hub for localized signal transduction. Mechanical, stress, or mitogen signals reach the nucleus and trigger the activation of several pathways, many effectors of which are processed at the NE. Therefore, the concept of the NE acting just as a barrier needs to be expanded to embrace all the dynamic processes that are indeed associated with it. In this context, dynamic protein association and turnover coupled to reversible post-translational modifications of NE components can provide important clues on how this integrated cellular machinery functions as a whole. Reversible protein phosphorylation is the most used mechanism to control protein dynamics and association in cells. Keys to the reversibility of the system are protein phosphatases and the regulation of their activity in space and time. As the NE is clearly becoming an interesting compartment for the control and transduction of several signalling pathways, in this review we will focus on the role of Protein Phosphatases at the NE since the significance of this class of proteins in this context has been little explored.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Resetting a functional G1 nucleus after mitosis.

The maintenance of the correct cellular information goes beyond the simple transmission of an intact genetic code from one generation to the next. Epigenetic changes, topological cues and correct protein-protein interactions need to be re-established after each cell division to allow the next cell cycle to resume in the correct regulated manner. This process begins with mitotic exit and re-sets all the changes that occurred during mitosis thus restoring a functional G1 nucleus in preparation for the next cell cycle. Mitotic exit is triggered by inactivation of mitotic kinases and the reversal of their phosphorylation activities on many cellular components, from nuclear lamina to transcription factors and chromatin itself. To reverse all these phosphorylations, phosphatases act during mitotic exit in a timely and spatially controlled manner directing the events that lead to a functional G1 nucleus. In this review, we will summarise the recent developments on the control of phosphatases and their known substrates during mitotic exit, and the key steps that control the restoration of chromatin status, nuclear envelope reassembly and nuclear body re-organisation. Although pivotal work has been conducted in this area in yeast, due to differences between the mitotic exit network between yeast and vertebrates, we will mainly concentrate on the vertebrate system.

Meeting report: mitosis and nuclear structure.

The Company of Biologists Workshop entitled 'Mitosis and Nuclear Structure' was held at Wiston House, West Sussex in June 2013. It provided a unique and timely opportunity for leading experts from different fields to discuss not only their own work but also its broader context. Here we present the proceedings of this meeting and several major themes that emerged from the crosstalk between the two, as it turns out, not so disparate fields of mitosis and nuclear structure. Co-chaired by Katherine Wilson (Johns Hopkins School of Medicine, Baltimore, MD), Timothy Mitchison (Harvard University, Cambridge, MA) and Michael Rout (Rockefeller University, New York, NY), this workshop brought together a small group of scientists from a range of disciplines to discuss recent advances and connections between the areas of mitosis and nuclear structure research. Several early-career researchers (students, postdoctoral researchers, junior faculty) participated along with 20 senior scientists, including the venerable and affable Nobel Laureate Tim Hunt. Participants were encouraged to embrace unconventional thinking in the 'scientific sandbox' created by this unusual combination of researchers in the inspiring, isolated setting of the 16th-century Wiston House.

DNA content of a functioning chicken kinetochore.

In order to understand the three-dimensional structure of the functional kinetochore in vertebrates, we require a complete list and stoichiometry for the protein components of the kinetochore, which can be provided by genetic and proteomic experiments. We also need to know how the chromatin-containing CENP-A, which makes up the structural foundation for the kinetochore, is folded, and how much of that DNA is involved in assembling the kinetochore. In this MS, we demonstrate that functioning metaphase kinetochores in chicken DT40 cells contain roughly 50 kb of DNA, an amount that corresponds extremely closely to the length of chromosomal DNA associated with CENP-A in ChIP-seq experiments. Thus, during kinetochore assembly, CENP-A chromatin is compacted into the inner kinetochore plate without including significant amounts of flanking pericentromeric heterochromatin.

Contrasting roles of condensin I and condensin II in mitotic chromosome formation.

In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-I-depleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.

Repo-man at the intersection of chromatin remodelling, DNA repair, nuclear envelope organization, and cancer progression.

Nuclear structure and chromatin changes are very useful biomarkers in cancer diagnosis. Despite this, their biological significance and relevance to cancer progression are still not well understood. The identification of new proteins that link the nuclear envelope to chromatin organization and the understanding of the molecular mechanisms underlying these connections have begun to provide some important clues. This review discusses the role of the nuclear protein Repo-Man (CDCA2) in the maintenance of genome stability. Repo-Man (CDCA2) is a targeting subunit for the protein phosphatase 1 involved in the dephosphorylation of histone H3 during mitotic exit. In this role, it is important for the chromatin organization in post-mitotic nuclei. Repo-Man (CDCA2) is also essential for proper nuclear envelope reformation and the regulation of DNA damage responses. The relevance of this complex for cancer biology is also corroborated by emerging evidence that provides a correlation between Repo-Man (CDCA2) expression levels and cancer progression; several studies now suggest that Repo-Man (CDCA2) represents a very strong prognostic marker for poor patient survival.

Cell Cycle-Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines.

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO). RIPPOs are tagged with the auxin-inducible degron (AID) or ascorbate peroxidase 2 (APEX2) modules, and PP1 substrate identification is conducted by SILAC proteomic-based approaches. Proteins in close proximity to RIPPOs are first identified through mass spectrometry (MS) analyses using the APEX2 system; then a list of differentially phosphorylated proteins upon RIPPOs rapid degradation (achieved via the AID system) is compiled via SILAC phospho-mass spectrometry. The "in silico" overlap between the two proteomes will be enriched for PP1 putative substrates. Several methods including fluorescence resonance energy transfer (FRET), proximity ligation assays (PLA), and in vitro assays can be used as substrate validations approaches.

Ki-67 is necessary during DNA replication for fork protection and genome stability.

BACKGROUND: The proliferation antigen Ki-67 has been widely used in clinical settings for cancer staging for many years, but investigations on its biological functions have lagged. Recently, Ki-67 has been shown to regulate both the composition of the chromosome periphery and chromosome behaviour in mitosis as well as to play a role in heterochromatin organisation and gene transcription. However, how the different roles for Ki-67 across the cell cycle are regulated and coordinated remain poorly understood. The progress towards understanding Ki-67 function have been limited by the tools available to deplete the protein, coupled to its abundance and fluctuation during the cell cycle. RESULTS: Here, we use a doxycycline-inducible E3 ligase together with an auxin-inducible degron tag to achieve a rapid, acute and homogeneous degradation of Ki-67 in HCT116 cells. This system, coupled with APEX2 proteomics and phospho-proteomics approaches, allows us to show that Ki-67 plays a role during DNA replication. In its absence, DNA replication is severely delayed, the replication machinery is unloaded, causing DNA damage that is not sensed by the canonical pathways and dependent on HUWE1 ligase. This leads to defects in replication and sister chromatids cohesion, but it also triggers an interferon response mediated by the cGAS/STING pathway in all the cell lines tested. CONCLUSIONS: We unveil a new function of Ki-67 in DNA replication and genome maintenance that is independent of its previously known role in mitosis and gene regulation.

Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis.

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex in certain experimental systems, but not in many others. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure. Here, live-cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 revealed that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the targeting subunit Repo-Man from recruiting protein phosphatase 1 (PP1) to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man-PP1 during anaphase. This activity, provisionally termed 'regulator of chromosome architecture' (RCA), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.

Mitotic chromosome condensation in vertebrates.

Work from several laboratories over the past 10-15 years has revealed that, within the interphase nucleus, chromosomes are organized into spatially distinct territories [T. Cremer, C. Cremer, Chromosome territories, nuclear architecture and gene regulation in mammalian cells, Nat. Rev. Genet. 2 (2001) 292-301 and T. Cremer, M. Cremer, S. Dietzel, S. Muller, I. Solovei, S. Fakan, Chromosome territories-a functional nuclear landscape, Curr. Opin. Cell Biol. 18 (2006) 307-316]. The overall compaction level and intranuclear location varies as a function of gene density for both entire chromosomes [J.A. Croft, J.M. Bridger, S. Boyle, P. Perry, P. Teague,W.A. Bickmore, Differences in the localization and morphology of chromosomes in the human nucleus, J. Cell Biol. 145 (1999) 1119-1131] and specific chromosomal regions [N.L. Mahy, P.E. Perry, S. Gilchrist, R.A. Baldock, W.A. Bickmore, Spatial organization of active and inactive genes and noncoding DNA within chromosome territories, J. Cell Biol. 157 (2002) 579-589] (Fig. 1A, A'). In prophase, when cyclin B activity reaches a high threshold, chromosome condensation occurs followed by Nuclear Envelope Breakdown (NEB) [1]. At this point vertebrate chromosomes appear as compact structures harboring an attachment point for the spindle microtubules physically recognizable as a primary constriction where the two sister chromatids are held together. The transition from an unshaped interphase chromosome to the highly structured mitotic chromosome (compare Figs. 1A and B) has fascinated researchers for several decades now; however a definite picture of how this process is achieved and regulated is not yet in our hands and it will require more investigation to comprehend the complete process. From a biochemical point of view a vertebrate mitotic chromosomes is composed of DNA, histone proteins (60%) and non-histone proteins (40%) [6]. I will discuss below what is known to date on the contribution of these two different classes of proteins and their co-operation in establishing the final mitotic chromosome structure.

A super-resolution map of the vertebrate kinetochore.

A longstanding question in centromere biology has been the organization of CENP-A-containing chromatin and its implications for kinetochore assembly. Here, we have combined genetic manipulations with deconvolution and super-resolution fluorescence microscopy for a detailed structural analysis of chicken kinetochores. Using fluorescence microscopy with subdiffraction spatial resolution and single molecule sensitivity to map protein localization in kinetochore chromatin unfolded by exposure to a low salt buffer, we observed robust amounts of H3K9me3, but only low levels of H3K4me2, between CENP-A subdomains in unfolded interphase prekinetochores. Constitutive centromere-associated network proteins CENP-C and CENP-H localize within CENP-A-rich subdomains (presumably on H3-containing nucleosomes) whereas CENP-T localizes in interspersed H3-rich blocks. Although interphase prekinetochores are relatively more resistant to unfolding than sur-rounding pericentromeric heterochromatin, mitotic kinetochores are significantly more stable, reflecting mitotic kinetochore maturation. Loss of CENP-H, CENP-N, or CENP-W had little or no effect on the unfolding of mitotic kinetochores. However, loss of CENP-C caused mitotic kinetochores to unfold to the same extent as their interphase counterparts. Based on our results we propose a new model for inner centromeric chromatin architecture in which chromatin is folded as a layered boustrophedon, with planar sinusoids containing interspersed CENP-A-rich and H3-rich subdomains oriented toward the outer kinetochore. In mitosis, a CENP-C-dependent mechanism crosslinks CENP-A blocks of different layers together, conferring extra stability to the kinetochore.